We have made an anchored RT-PCR-based cDNA library from 14 day fetal thymii cultured in deoxyguanosine and treated with anti-CD45 antibodies to deplete the population of hematopoietic cells. The cDNA library was substracted with polyA plus RNA prepared from a fibroblast cell line and from whole spleen. Random 3' sequencing of 288 cDNAs revealed that 182 were not in the databases of known sequences. The novel cDNAs were then screened by Northern blotting for expression in various tissues or in a set of SV40 transformed thymic epithelial cell lines developed by Dr. Barbara Knowles. Genes expressed selectively in the thymus or selectively in one of the stromal cell lines were completely sequenced. So far we have identified two interesting genes. One encodes a 12 transmembrane spanning helix-containing protein that is distantly related to the Na+/H+ antiporter family and is only expressed in thymus. It is 10-fold enriched in SCID-thymus. The second encodes a membrane bound serine protease that is expressed in one cortical thymic epithelial cell line and activated T and B cells. The protease domain is linked to the membrane by a series of LDL-like domains. Currently, we are attempting to raise antibodies against both these proteins in order to study their function in thymic development.